P1341: LC3-ASSOCIATED PHAGOCYTOSIS IS IMPAIRED IN BONE MARROW MACROPHAGES DURING AGEING LEADING TO DEFECTIVE PHAGOCYTOSIS AND ACCUMULATION OF APOPTOTIC DEBRIS WITHIN THE BONE MARROW.
Authors: Katherine Hampton, Charlotte Hellmich, Rebecca Maynard, Aisha Jibril, Jamie Moore, Kristian Bowles, Stuart Rushworth
Journal: HemaSphere (2023)

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: During ageing, the phagocytic function of macrophages declines, negatively impacting immune response, healthy tissue homeostasis and inflammation. Mechanistically, phagocytosis by macrophages occurs either dependently or independently of LC3, creating an anti-inflammatory or pro-inflammatory response. Specifically, LC3-associated phagocytosis (LAP) is physiologically triggered by pathogens and cellular debris via interaction with phagocyte surface receptors. Aims: Here, we investigate whether age-related macrophage functional impairment is associated with LC3. Methods: Bone marrow (BM) was isolated from young (8-20 weeks) and aged (18-24 months) C57Bl/6 and LAP-deficient (Atg16lE230–) mice, and macrophages were analysed using flow cytometry for M1 and M2 phenotypes. To establish their phagocytic function, cultured bone marrow derived macrophages (BMDM) were treated with pHrodo E. coli bioparticles for two hours, before being fixed, stained for LC3 and imaged via immunofluorescent microscopy. Results: Aged WT mice had a 2-fold increase in macrophage numbers compared to young WT, the majority of which are polarised with M2-like characteristics. Paradoxically, despite increased macrophage numbers, aged WT mice had significantly increased apoptotic cells and cellular debris within their BM compared to young WT mice. However, both in vivo and in vitro, there was no significant difference in total phagocytic potential between aged and young mice, measured by the proportion of macrophages with uptake pHrodo E. coli bioparticles. However aged WT BM macrophages did not recruit LC3 during phagosome formation, whereas macrophages from young animals LC3 was recruited. Consequently, macrophages from older animals accumulated large amounts of E. coli bioparticles and apoptotic bodies when compared to young macrophages. This suggests defective processing of the E. coli bioparticles by the phagosome once the phagosome has formed. Next, we compared this aged phenotype seen to young LAP-/- mice. LAP-/- mice also had increased macrophages within the BM, and a greater proportion expressed M2-like characteristics. Furthermore, similarly to aged WT mice, LAP-/- animals had an accumulation of cellular debris and apoptotic cells in their BM compared to young WT mice. Finally, cultured BMDM from LAP-/- mice also accumulated a greater proportion of pHrodo E. coli bioparticles, indicating that they too, were less able to process the E. coli and thus had impaired phagocytic function. Therefore, LAP-/- mice display a similar phenotype to that seen in the aged WT mice. Summary/Conclusion: To conclude, here we show that during ageing, macrophage number increases. However, aged macrophages have a defective phagocytic function, in part due to a failure of LC3-mediated phagosome clearance, thus resulting in the accumulation of particles within the phagosome with little LC3-association. This ageing phenotype is also seen in young LAP-/- mice, providing strong evidence that loss of LC3 is at least in part responsible for this age-related decline in macrophage function. Keywords: Macrophage, Phagocytosis, Aging